Šíchová et al. BMC Evolutionary Biology (2015) 15:89
DOI 10.1186/s12862-015-0375-4
RESEARCH ARTICLE
Open Access
Dynamic karyotype evolution and unique sex
determination systems in Leptidea wood
white butterflies
Jindra Šíchová1,2, Anna Voleníková2, Vlad Dincă3,4, Petr Nguyen1,2, Roger Vila4, Ken Sahara5 and František Marec1,2*
Abstract
Background: Chromosomal rearrangements have the potential to limit the rate and pattern of gene flow within
and between species and thus play a direct role in promoting and maintaining speciation. Wood white butterflies
of the genus Leptidea are excellent models to study the role of chromosome rearrangements in speciation because
they show karyotype variability not only among but also within species. In this work, we investigated genome
architecture of three cryptic Leptidea species (L. juvernica, L. sinapis and L. reali) by standard and molecular
cytogenetic techniques in order to reveal causes of the karyotype variability.
Results: Chromosome numbers ranged from 2n = 85 to 91 in L. juvernica and 2n = 69 to 73 in L. sinapis (both from
Czech populations) to 2n = 51 to 55 in L. reali (Spanish population). We observed significant differences in
chromosome numbers and localization of cytogenetic markers (rDNA and H3 histone genes) within the offspring of
individual females. Using FISH with the (TTAGG)n telomeric probe we also documented the presence of multiple
chromosome fusions and/or fissions and other complex rearrangements. Thus, the intraspecific karyotype variability
is likely due to irregular chromosome segregation of multivalent meiotic configurations. The analysis of female
meiotic chromosomes by GISH and CGH revealed multiple sex chromosomes: W1W2W3Z1Z2Z3Z4 in L. juvernica,
W1W2W3Z1Z2Z3 in L. sinapis and W1W2W3W4Z1Z2Z3Z4 in L. reali.
Conclusions: Our results suggest a dynamic karyotype evolution and point to the role of chromosomal
rearrangements in the speciation of Leptidea butterflies. Moreover, our study revealed a curious sex determination
system with 3–4 W and 3–4 Z chromosomes, which is unique in the Lepidoptera and which could also have played
a role in the speciation process of the three Leptidea species.
Keywords: Lepidoptera, Wood white butterflies, Karyotype variability, Fluorescence in situ hybridization
Chromosome fusion and fission, Multiple sex chromosomes, Speciation
Background
Speciation, i.e. the origin of new species, is a complex
evolutionary process which leads to the formation of
barriers preventing gene flow between emerging species.
Defining the factors that generate such barriers is a central goal for evolutionary biologists. Among animals,
moths and butterflies (insect order Lepidoptera) represent an ideal model group for the study of various
* Correspondence: [email protected]
1
Institute of Entomology, Biology Centre CAS, 370 05 České Budějovice,
Czech Republic
2
Faculty of Science, University of South Bohemia, 370 05 České Budějovice,
Czech Republic
Full list of author information is available at the end of the article
aspects of speciation. This is mainly due to the immense
diversity of Lepidoptera, which include nearly 160,000
species and belong to the most speciose groups of animals [1]. Moreover, the study of moths and butterflies
provides a number of practical advantages. Many species
can be easily collected in the field, reared and hybridized
in laboratory conditions and experiments can be replicated fairly often due to the relatively short generation
time of many species.
Among traditional models, the Heliconius butterflies
have been the subject of a high number of evolutionary
studies showing that various wing patterns, resulting
from predator-induced selection through Müllerian
mimicry, ultimately lead to divergence and speciation
© 2015 Šíchová et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Šíchová et al. BMC Evolutionary Biology (2015) 15:89
(e.g. [2–6]). The shift in colour pattern mimicry also
played a key role in generating pre-mating isolation as
male mate preferences often led to strong assortative
mating between individuals with similar wing pattern
phenotypes [7]. Swallowtail butterflies of the genus Papilio are another diverse group of Lepidoptera where the
evolution of mimicry greatly contributed to their spectacular radiation [8–10]. Lepidoptera also include
models for research of sex pheromone communication
and its role as a pre-zygotic barrier [11, 12]. A wellknown example is the European corn borer, Ostrinia
nubilalis, which comprises two sympatric races that are
prevented from mating by utilizing opposite sex pheromone isomers of the same compound [13, 14]. The
butterfly subgenus Agrodiaetus is one of the few taxa
where reinforcement of pre-zygotic isolation has been
demonstrated [15].
It is generally accepted that chromosomal rearrangements have the potential to limit introgression and thus
facilitate the development and maintenance of reproductive isolation by means of suppressed recombination
[16–18]. Reduced recombination enables the accumulation of genetic incompatibilities and leads to divergence
and speciation. Suppression of recombination is an intrinsic feature of sex chromosomes which were suggested to play a disproportionate role in lepidopteran
speciation [19, 20]. Recent studies in geographic subspecies of wild silkworms, Samia cynthia ssp. (Saturniidae),
suggest that chromosomal rearrangements resulting in
multiple sex chromosomes may also contribute to the
formation of reproductive barriers and thus promote divergence and eventually speciation [21, 22]. Moreover,
the holokinetic nature of lepidopteran chromosomes, i.e.
the lack of a distinct primary constriction (the centromere), is expected to facilitate karyotype evolution
mainly via chromosomal fusion and fission by reducing
the risk of formation of dicentric and acentric chromosomes [23]. However, results of comparative genomics
revealed a high degree of conserved synteny of genes between the silkworm Bombyx mori (Bombycoidea) and
several other lepidopteran species [24–30]. The extensive
conservation of chromosome print across Lepidoptera
suggests evolutionary stability of lepidopteran karyotypes,
with most of haploid chromosome numbers ranging n =
28–32 [31] and the most common and probably also
ancestral number of n = 31 [30, 32].
The remarkably stable chromosome numbers and
highly conserved synteny of genes between chromosomes of distant species contrast with the exceptional
diversity of karyotypes found in some lepidopteran taxa.
Probably the greatest interspecific karyotype variation in
the animal kingdom was found in blue butterflies
(Lycaenidae: Polyommatinae) of the genus Polyommatus
with haploid chromosome numbers ranging from n = 10
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to n = 223 [15, 33–35]. The latter, observed in the Atlas
blue, Polyommatus atlantica, represents the highest
chromosome number not only of Lepidoptera but of all
animals [36]. In addition, blue butterflies of the subgenus Agrodiaetus represent the group with the largest
difference in the number of chromosomes between sister
species. Karyotypes of Polyommatus (Agrodiaetus) biruni
and P. (A.) posthumus consist of n = 10 and n = 90 elements, respectively, with no intermediate karyomorphs.
The similarity in genome size of these closely related
species suggests that the karyotype variation is not
caused by polyploidy but arose through chromosomal
rearrangements such as fusion and fission [37]. However,
recent comparative phylogenetic studies found little evidence supporting the role of chromosomal rearrangements in the speciation of Agrodiaetus blues and rather
stressed the importance of reinforcement of their prezygotic isolation [15].
Exceptional intraspecific variability of karyotypes was
also found in wood white butterflies of the genus Leptidea comprising several Eurasian species [38, 39]. In this
genus, chromosome numbers vary greatly between and
within species. While two species with predominantly
Eastern Palaearctic distribution, L. morsei and L. amurensis, probably have a constant number of chromosomes
(n = 54 and n = 61, respectively; [40]), three cryptic species mainly from the Western Palaearctic have a variable
number of chromosomes [38, 41]. The most striking
case is L. sinapis, which shows a gradual decrease in the
diploid chromosome number from 2n = 106 in Spain to
2n = 56 in eastern Kazakhstan, resulting in a 6000 km
wide chromosomal cline of recent origin [39]. Excluding
polyploidy, this is the widest known within-species
chromosome number range for any animal or plant.
Moreover, a variable number of chromosomes was
described in the other two cryptic species, L. reali (2n =
52–54) and L. juvernica (2n = 80–84) [38].
Although the nature of dynamic evolution of Leptidea
karyotypes and its role in speciation is not yet known,
the chromosomal cline found in L. sinapis provided
strong evidence for rapid and extensive within-species
accumulation of numerous chromosomal rearrangements [39]. While such clinal speciation is theoretically
possible, it is difficult to document without further research. In this study, we integrated standard cytogenetic
techniques and FISH (fluorescence in situ hybridization)
mapping of major ribosomal RNA (rRNA) and H3 histone genes to study among- and within-species variability in the karyotypes of three cryptic Leptidea species
(L. juvernica, L. sinapis and L. reali). We also determined the sex chromosome constitution using genomic
in situ hybridization (GISH) and examined molecular
differentiation of the sex chromosomes through comparative genomic hybridization (CGH). Cytogenetic
Šíchová et al. BMC Evolutionary Biology (2015) 15:89
characteristics were compared with the aim of understanding karyotype and sex chromosome evolution in
Leptidea butterflies.
Results
Molecular identification of Leptidea specimens
Morphometric analysis of genitalia allowed us to identify
only two groups, L. sinapis and the group consisting of
L. reali and L. juvernica, whose genitalia cannot be reliably distinguished [38]. Phylogenetic analyses based on
two DNA markers, the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and the nuclear internal transcribed spacer 2 (ITS2) sequence, revealed three supported
major clades corresponding to L. juvernica, L. sinapis and
L. reali (Fig. 1 and Additional file 1: Figure S1). Relationships among these clades coincide with previous results
[38, 41] with L. juvernica being sister to the species pair
L. sinapis and L. reali.
Karyotype differences in chromosome number and
structure
Chromosome numbers of all three Leptidea species were
counted from mitotic metaphase complements prepared
from wing imaginal discs of the last instar larvae and
stained by means of FISH with (TTAGG)n telomeric
probes (tel-FISH) to facilitate identification of individual
chromosome elements. In each Leptidea species, several
tens of mitotic metaphases were analysed in the progeny
of individual females.
Based on repeated counts we found that chromosome
numbers differ considerably in all three species studied.
Moreover, we observed differences in the number of
chromosomes even among the offspring of individual females. We established that, in the population studied,
the chromosome number is not fixed and ranges from
2n = 85 to 91 in L. juvernica and 2n = 69 to 73 in L. sinapis (both from Czech populations) to 2n = 51 to 55 in
L. reali (Spanish population). Mitotic complements of
L. juvernica and L. sinapis also displayed a higher variability in chromosome size, having mostly middle- or
small-sized chromosomes (L. juvernica, Fig. 2a) or a mixture of large- and small-sized chromosomes (L. sinapis,
Fig. 3c, d, f), while in L. reali we observed larger chromosomes of a similar size (Fig. 2b).
In male meiotic metaphase I (MI) and pachytene complements of all studied species we observed complex
chromosomal rearrangements (Fig. 2c, d) and conspicuous heterochromatin blocks highlighted with DAPI
(Fig. 2d). However, in female pachytene complements
these DAPI-positive blocks did not allow the identification
of a sex chromosome bivalent according to the W
chromosome, which is usually the only largely heterochromatic element present in lepidopteran karyotypes [42, 43].
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Chromosomal location of major rDNA
FISH with the biotin-labelled 18S ribosomal DNA
(rDNA) probe combined with the digoxigenin-labelled
(TTAGG)n telomeric probe did not reveal any difference
in the number of major rDNA clusters in offspring of
three L. juvernica and three L. reali females. In both species, the rDNA probe mapped to two mitotic metaphase
chromosomes of a similar size (Fig. 3a, b; for simplification, hybridization signals of the telomeric probe are not
shown) and to a single bivalent in the pachytene stage
(insets of Fig. 3a, b). This clearly indicates the presence
of a single pair of chromosomes, each carrying a cluster
of rRNA genes forming a nucleolar organizer region
(NOR). However, we found a substantial interspecific
difference in the location of rDNA. While the NORbivalent in pachytene nuclei of L. juvernica showed the
cluster of rRNA genes associated with a large interstitial
block of DAPI-positive heterochromatin (inset of Fig. 3a),
rDNA occupied a large terminal segment of the NORbivalent in L. reali. In the latter species, rDNA was not
associated with heterochromatin, which was observed at
the opposite end of the NOR-bivalent (inset of Fig. 3b).
In L. sinapis, we found intraspecific variability in the
number and position of rDNA clusters both within and
among the offspring of individual females (Fig. 3c-g;
hybridization signals of the telomeric probe are not
shown). In mitotic metaphase complements from the
offspring of one female the 18S rDNA probe localized
four rDNA sites at the ends of four middle-sized chromosomes (Fig. 3c), thus indicating two pairs of NORchromosomes. However, in mitotic metaphases from the
offspring of another female we found either two terminal
and one interstitial signal (Fig. 3d) or three terminal
hybridization signals (Fig. 3f ). The difference among siblings was confirmed in pachytene nuclei where we observed two hybridization signals in a trivalent and one in
an element of a bivalent that was heterozygous for
rDNA (Fig. 3e) or a pair of signals in a small bivalent
and one signal in a larger element of another bivalent
(Fig. 3g), respectively.
Chromosomal location of H3 histone genes
FISH with the H3 histone probe combined with tel-FISH
showed constant results only in L. reali. In all examined
larvae from progenies of three different females we found
one interstitial cluster of H3 histone genes in a large
pachytene bivalent and two clusters in mitotic metaphase
complements (Fig. 4a; hybridization signals of the telomeric probe are not shown). In the pachytene bivalent, the
H3 cluster was localized next to a small block of DAPIpositive heterochromatin (inset of Fig. 4a).
In L. sinapis and L. juvernica, we observed intraspecific variability in the number and location of H3 histone
gene clusters both within and among offspring of
Šíchová et al. BMC Evolutionary Biology (2015) 15:89
Fig. 1 (See legend on next page.)
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Šíchová et al. BMC Evolutionary Biology (2015) 15:89
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(See figure on previous page.)
Fig. 1 Neighbor-joining tree of mitochondrial COI haplotypes of L. sinapis (grey background), L. reali (orange background) and L. juvernica
(blue background). Specimens sequenced and analysed in this study are indicated by an asterisk and were combined with representatives of all
available haplotypes of L. sinapis, L. reali and L. juvernica identified in a previous study [41]. Leptidea amurensis, L. lactea, L. morsei and L. duponcheli
were used as outgroup. For the origin of all specimens and GenBank accession numbers, see Additional file 5: Table S1. The scale represents 0.01
substitutions per site. Bootstrap supports (100 replicates) are shown next to the recovered nodes. Representative male specimens and genitalia
(drawn to scale, with phallus and saccus indicated) are shown
Fig. 2 Karyotype analysis of mitotic and meiotic chromosomes of Leptidea species by FISH with the (TTAGG)n telomeric probe. Hybridization signals of
the Cy3-dUTP-labelled telomeric probe (red) indicate chromosome ends in (a–c). Chromosomes were counterstained with DAPI (blue). White arrows
indicate chromosome multivalents and asterisks show heterochromatic blocks. a Mitotic metaphase of L. juvernica female with numerous middle- or
small-sized chromosomes (2n = 85). b Mitotic metaphase of L. reali female with large chromosomes of a similar size (2n = 55). c Meiotic metaphase I of
L. juvernica male showing several chromosome multivalents. d Meiotic pachytene complement of L. juvernica female showing several chromosome
multivalents and numerous blocks of DAPI-highlighted heterochromatin. Scale bars = 10 μm; (a) and (b) have the same scale
Šíchová et al. BMC Evolutionary Biology (2015) 15:89
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Fig. 3 Localization of rDNA clusters in spread chromosome preparations of three Leptidea species by FISH with 18S rDNA probe. Chromosomes
were counterstained with DAPI (blue). Arrows show hybridization signals of the 18S rDNA probe (red); asterisks indicate DAPI-positive blocks of
heterochromatin. a Mitotic metaphase of L. juvernica male (2n = 90); the inset in the upper right corner shows the pachytene NOR-bivalent with a large
interstitial rDNA cluster. b Mitotic metaphase of L. reali female (2n = 52); the inset in the upper right corner shows the pachytene NOR-bivalent with a
large terminal rDNA cluster. c Male mitotic metaphase (2n = 69) with a typical hybridization pattern found in the offspring of one L. sinapis female.
Figures (d–g) show a variable pattern in the offspring of another L. sinapis female: (d) mitotic metaphase of male offspring (2n = 71); (e) hybridization
signals on pachytene chromosomes of the same male offspring (schematic drawing shows the structure of a trivalent carrying two out of three rDNA
clusters); (f) mitotic metaphase of female offspring (2n = 73); (g) hybridization signals on pachytene chromosomes of the same female offspring
(schematic drawing shows the structure of a bivalent heterozygous for a terminal rDNA cluster). Scale bar = 10 μm
individual females (Fig. 4b-i; hybridization signals of the
telomeric probe are not shown). In some offspring of
one L. sinapis female we observed an interstitial cluster
of H3 histone genes in a long pachytene bivalent (inset
of Fig. 4b) corresponding to two hybridization signals in
a pair of mitotic chromosomes (Fig. 4b) like in L. reali.
However, in other offspring of the same female, a single
H3 histone gene array mapped to a subterminal region
of a short pachytene bivalent (inset of Fig. 4c), corresponding to terminal hybridization signals in a pair of
small mitotic chromosomes (Fig. 4c). In the offspring of
another L. sinapis female, hybridization signals positioned two H3 gene clusters to a pachytene trivalent,
one terminal in a short chromosome and the other
interstitial in a long chromosome (Fig. 4d). In accordance with this hybridization pattern, the H3 probe
identified two mitotic chromosomes, one small and one
large (Fig. 4e). In pachytene nuclei of L. juvernica, the
H3 probe hybridized most often to a tetravalent. We
found three clusters of hybridization signals, one terminal in a short element and two interstitial in two long
elements of the tetravalent (Fig. 4g). The hybridization
pattern was confirmed in mitotic nuclei, where the
probe mapped H3 gene arrays to the end of a small
chromosome and to the middle of two larger chromosomes (Fig. 4f ). The number and location of H3 gene
clusters was characteristic for the offspring of four
L. juvernica females. However, in the offspring of another female we found an additional (fourth) H3 gene
cluster located at the end of one element of a pachytene
bivalent (Fig. 4i), corresponding to a total number of
four hybridization signals in mitotic nuclei (Fig. 4h).
Šíchová et al. BMC Evolutionary Biology (2015) 15:89
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Fig. 4 Localization of H3 histone gene clusters in spread chromosome preparations of three Leptidea species by FISH with H3 gene probe.
Chromosomes were counterstained with DAPI (blue). Arrows indicate hybridization signals of the H3 probe (red); asterisks show DAPI-positive
blocks of heterochromatin. a Mitotic metaphase of L. reali female; the inset in the upper right corner shows the pachytene H3 cluster-carrying
bivalent. Figures (b–e) show intraspecific variability in the location of H3 histone gene clusters in L. sinapis: (b) mitotic metaphase of male larva;
the inset shows the pachytene bivalent carrying a cluster of H3 genes; (c) mitotic metaphase of another male from the same offspring; the inset
shows the pachytene bivalent carrying a cluster of H3 genes; (d) pachytene trivalent observed in the female offspring of another female
(schematic drawing shows the structure of the trivalent and positions of two H3 clusters); (e) female mitotic metaphase of the same individual.
Figures (f–i) show intraspecific variability in the location of H3 histone gene clusters in L. juvernica: (f) male mitotic metaphase with three
hybridization signals, observed in the vast majority of L. juvernica larvae; (g) pachytene tetravalent of the same individual (schematic drawing
shows the structure of the tetravalent and positions of three H3 clusters); (h) mitotic metaphase with four hybridization signals found in one male
offspring of another female; (i) pachytene tetravalent with three hybridization signals (see schematic drawing in g) and bivalent with the fourth
hybridization signal located at the end of one homologue. Scale bars = 10 μm; except for (f) all images have the same scale
Sex chromosome constitution
We first examined the presence or absence of female
specific sex chromatin in polyploid somatic nuclei of all
three Leptidea species. The sex chromatin consists of
multiple copies of the W chromosome, which usually
form one conspicuous heterochromatin body in somatic
interphase nuclei of lepidopteran females [44]. In the
majority of female larvae of all three species, we observed one larger, more intensely stained heterochromatin body and two tiny indistinct bodies (Additional file 2:
Figure S2a). Yet the larger body was much smaller in
comparison to the sex chromatin typically observed in
Šíchová et al. BMC Evolutionary Biology (2015) 15:89
females of other lepidopteran species (cf. [45, 46]). In
other females we found a variable number of tiny heterochromatin bodies ranging from none to four. Similar
findings were made in branched nuclei of adult females
with a higher level of ploidy (Additional file 2: Figure
S2b). In the majority of Leptidea males, no sex chromatin was observed in polyploid cells (Additional file 2:
Figure S2c). However, in a few male specimens we found a
tiny heterochromatin body of uncertain origin (Additional
file 2: Figure S2d). The small size and fragmentation of sex
chromatin in Leptidea females indicate the presence of interchromosomal rearrangements involving the W chromosome (see [42, 47]).
To identify the W chromosome we examined spread
preparations of pachytene oocytes using a combination
of GISH and tel-FISH. While GISH differentiated the W
chromosome thread in female pachytene nuclei, the telomeric probe helped us to determine chromosomal ends.
The female-derived genomic probe also hybridized to
heterochromatin blocks on autosomes, which made the
identification of the W chromosome more difficult in
pachytene nuclei and impossible in mitotic metaphases.
Nevertheless, the analysis revealed multiple sex chromosomes in all three Leptidea species with 3–4 W and 3–4 Z
chromosomes (Fig. 5a–l; hybridization signals of the telomeric probe are shown in Additional file 3: Figure S3a–l).
In L. juvernica, we observed a W1W2W3Z1Z2Z3Z4 sex
chromosome constitution (Fig. 5a–d). While the female
genomic DNA (gDNA) probe strongly bound to two W
chromosomes, the third one was highlighted only partially
(Fig. 5c). Moreover, two of the three W chromosomes
were partially differentiated by DAPI-positive heterochromatin (Fig. 5b, two upper arrows). We found only a small
heterochromatin block at the very end of the third W
chromosome (Fig. 5b, the lower arrow). In L. sinapis, we
found a W1W2W3Z1Z2Z3 sex chromosome system
(Fig. 5e–h) with an intensely stained block of heterochromatin on one of the W chromosomes (Fig. 5f, the middle
arrow). We also found a small heterochromatin block at
the very end of the smallest W chromosome (Fig. 5f, the
upper arrow) while the third W chromosome was discernible only due to hybridization signals of the female gDNA
probe (Fig. 5g). In the third species, L. reali, the sex
chromosome constitution was W1W2W3W4Z1Z2Z3Z4
(Fig. 5i–l). Except for the smallest W, the W chromosomes
were highlighted with the female gDNA probe (Fig. 5k),
but the staining pattern of DAPI was indistinctive with
only few small heterochromatin blocks of higher intensity
(Fig. 5j, arrows).
The level of molecular differentiation of the W and Z
chromosomes was examined using CGH. In pachytene
oocytes of the three Leptidea species, the WZ multivalent was discernible from autosomes due to stronger
binding of both female- and male-derived probes to the
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W chromosomes (Fig. 6a–o). A detailed analysis of the
WZ multivalent at the pachytene stage of L. juvernica
revealed a similar labelling pattern of both probes
(Fig. 6a–e). In L. sinapis, the W chromosomes were decorated with strong but scattered hybridization signals of
both genomic probes (Fig. 6f, h, i) with a slight preference for the female probe (Fig. 6h). The highest level of
molecular differentiation of the W and Z chromosomes
was observed in L. reali (Fig. 6k–o), where three out of
four W chromosomes were preferentially labelled by the
female-derived probe (Fig. 6m). However, the smallest
W chromosome was almost indistinguishable from the Z
chromosome (Fig. 6m–o). Hybridization signals of the
male-derived genomic probe were considerably weaker,
except for a few intense heterochromatin blocks located
on one W chromosome (Fig. 6n).
Discussion
We performed a detailed karyotype analysis of three
cryptic Leptidea species (L. juvernica, L. sinapis and
L. reali) by means of standard and molecular cytogenetic
techniques. Previous studies showed both inter- and intraspecific variation in chromosome numbers in all three
studied species. However, the results were based on
chromosome counts from squash preparations of metaphase I spermatocytes [38, 39], which did not allow the
analysis of complex meiotic figures such as multivalents.
Using FISH with (TTAGG)n telomeric probes, we confirmed the presence of numerous multivalents in female
pachytene nuclei as well as in male pachytene and metaphase I complements in all three species. Detailed analysis of male and female mitotic metaphases prepared
from wing imaginal discs allowed us to determine more
accurately the range of diploid chromosome numbers
that were 2n = 51–55 in L. reali (Spanish population),
2n = 69–73 in L. sinapis and 2n = 85–91 in L. juvernica
(both Czech populations). These numbers are broadly in
line with previous findings [38, 39]. Nevertheless, we further extended the range of chromosome numbers observed in L. juvernica and L. reali and provided data
from new localities for L. juvernica and L. sinapis.
Besides inter- and intraspecific karyotype variability, the
analysis of mitotic chromosomes allowed us to identify differences in chromosome numbers even within offspring of
individual females of all three species. Although such intrapopulation variability could be caused by the presence of
supernumerary chromosomes, i.e. B-chromosomes, this
scenario was deemed unlikely in the case of Leptidea species [39]. Yet, we cannot totally exclude the involvement of
B-chromosomes, especially as they were observed in related white butterflies from the family Pieridae [34]. In wild
silkworms (Samia cynthia), chromosomal polymorphism
among geographical populations/subspecies was ascribed
to repeated autosome-sex chromosome fusions resulting in
Šíchová et al. BMC Evolutionary Biology (2015) 15:89
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Fig. 5 Genomic in situ hybridization (GISH) in pachytene oocytes of Leptidea juvernica (a–d), L. sinapis (e–h) and L. reali (i–l). Female-derived
genomic probes were labelled with fluorescein-12-dUTP (green) and chromosomes were counterstained with DAPI (blue). Figures (a–d), (e–h)
and (i–l) show detailed analyses of sex chromosome multivalents W1-nZ1-n: (a, e, i) merged images of female genomic probes and DAPI staining;
(b, f, j) DAPI images; arrows indicate DAPI-positive W-chromosome segments and heterochromatic blocks at the end of the W chromosomes;
(c, g, k) hybridization pattern of the female genomic probes; the asterisk indicates an undifferentiated segment of one of the W chromosomes;
(d, h, l) schematic drawings of the sex chromosome multivalents. Scale bar = 10 μm
neo-sex chromosomes and reduced chromosome numbers [21]. Similar intraspecific variation resulting in reduced chromosome numbers was also reported in
grasshoppers (e.g. [48]) and mammals (e.g. [49, 50]),
mainly as a result of Robertsonian translocations.
However, karyotype variation observed in the three
Leptidea species surpasses previous reports. Our findings, namely the relatively low number of large chromosomes in L. reali, variability in chromosome size in the
other two species and the occurrence of multivalents in
meiotic nuclei of all three species, suggest that the Leptidea karyotypes are differentiated by multiple chromosome fusions and fissions. In addition, our preliminary
data showing a similar size of interphase nuclei in the
Leptidea species studied (Additional file 4: Figure S4)
suggest that their karyotypes did not differentiate
through polyploidy. Uneven chromosome segregation
of multivalents during meiotic division is thus the most
plausible explanation for the intraspecific and intrapopulation karyotype variations.
Šíchová et al. BMC Evolutionary Biology (2015) 15:89
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Fig. 6 Comparative genomic hybridization (CGH) in pachytene oocytes of Leptidea juvernica (a–e), L. sinapis (f–j) and L. reali (k–o). Female-derived
genomic probes were labelled with fluorescein-12-dUTP (green), male-derived genomic probes were labelled with Cy3-dUTP (red) and chromosomes
were counterstained with DAPI (blue). Figures (a–e), (f–j) and (k–o) show detailed analyses of sex chromosome multivalents W1-nZ1-n: (a, f, k) merged
images of both genomic probes and DAPI staining; (b, g, l) DAPI images; (c, h, m) female genomic probes; arrows indicate W-chromosome segments
with female-specific hybridization pattern; (d, i, n) male genomic probes; (e, j, o) schematic drawings of the sex chromosome multivalents.
Scale bars = 10 μm
The karyotypes of Leptidea species also differed in the
number and location of two cytogenetic markers used in
this study, clusters of rRNA genes (major rDNA) and
H3 histone genes. In L. reali, the species with the lowest
chromosome number, all larvae showed consistent results with a single terminal rDNA cluster and an interstitial cluster of H3 genes per haploid genome. The
highest variability in the number and position of both
cytogenetic markers was observed within and among the
offspring of individual L. sinapis females. In L. juvernica,
the number and location of H3 histone genes differed in
the progeny of individual females, while one rDNA cluster was always located in the middle of one pachytene
bivalent. Except for L. reali, both cytogenetic markers
often marked multivalents. Interspecific differences as
well as intrapopulation variability in rDNA distribution
Šíchová et al. BMC Evolutionary Biology (2015) 15:89
in L. sinapis are in agreement with the hypothesis on
dynamic evolution of genes for major RNAs in Lepidoptera [51]. However, the differences in the number and
location of H3 histone genes in L. sinapis and especially
L. juvernica are rather surprising, since this marker
shows a highly conserved pattern in the lepidopteran
family Tortricidae [52] and other insect groups, such as
the Acrididae grasshoppers [53] and Scarabaeinae beetles [54]. Our results thus support the previously
reported intraspecific variability in the karyotype of
L. sinapis [39] and highlight the ongoing explosive
karyotype evolution in all three Leptidea species.
To further explore Leptidea karyotypes and to identify
their sex chromosomes we performed GISH combined
with tel-FISH. In pachytene nuclei of lepidopteran females, the WZ bivalent is usually easily discernible with
GISH by deep staining of the W chromosome thread with
the fluorescently labelled female gDNA probe [43, 45, 55].
In all three Leptidea species, the analysis revealed unique
sex chromosome systems with the following constitutions:
W1W2W3Z1Z2Z3Z4 in L. juvernica, W1W2W3Z1Z2Z3 in
L. sinapis and W1W2W3W4Z1Z2Z3Z4 in L. reali. The constitution seemed to be stable in the progenies of individual
females. Multiple sex chromosome systems have been
documented in mammals [56, 57], fish [58, 59] and spiders
[60, 61]. However, the majority of moths and butterflies
show a WZ/ZZ sex chromosome system. Multiple sex
chromosomes have so far been found only in seven genera
and only in two different constitutions, either with
W1W2Z or WZ1Z2 trivalents in females [46]. Thus, the
sex chromosome constitutions observed in Leptidea stand
out for its complexity and the number of chromosomes
involved in the multivalent in meiosis. In addition, this is
the first case of multiple sex chromosomes in butterflies
(Papilionoidea). Besides sex chromosomes, the gDNA
probes also highlighted heterochromatin blocks abundantly present in the karyotypes of all three studied species. In Lepidoptera, heterochromatin is usually confined
to the NOR containing rDNA repeats [51, 52] and to the
W chromosome [44, 62]. So far, similar heterochromatin
blocks have been found only in subtelomeric regions of
the white butterfly Pieris brassicae (Pieridae) [51] and on
chromosome 24 of Bombyx mori [63]. In Leptidea, however, heterochromatin was evenly distributed throughout
the whole genome. Such distribution suggests the preponderance of tandemly arranged repetitive sequences in
Leptidea genomes in comparison with other lepidopteran
species, which could ultimately contribute to the remarkable karyotype diversity in this group.
In addition to the different sex chromosome constitution, the three Leptidea species also differed in their
overall genomic hybridization pattern. In L. sinapis and
L. juvernica, all W chromosomes were at least partially
differentiated by strong binding of fluorescently labelled
Page 11 of 16
gDNA probes with GISH and CGH, indicating the accumulation of repetitive sequences and transposable elements in the W chromosomes (cf. [64]). In L. reali, one
of the W chromosomes was not highlighted by any
gDNA probe. This W chromosome probably represents
an evolutionarily young element, which did not have sufficient time to differentiate. Individual Z chromosomes
involved in multivalents thus probably correspond to the
so-called evolutionary strata, which were also reported
in mammals, birds and plants [65–69]. Moreover, similar
hybridization patterns of male and female genomic probes
in CGH experiments suggest a predominance of common
repetitive sequences and transposons and a low amount of
W-specific sequences on the W chromosomes of L. sinapis and L. juvernica. On the contrary, the preferential
binding of the female-derived genomic probe to three of
the four W chromosomes in L. reali suggest a relatively
high proportion of W-specific sequences.
Observed differences in chromosome numbers and location of the major rDNA and H3 histone gene clusters
as well as the existence of complex sex chromosome systems corroborate the role of chromosomal rearrangements in the speciation of the closely related Leptidea
species examined in this study. It has been shown that
chromosomal rearrangements have a potential to limit
gene flow and thus facilitate the development and maintenance of reproductive isolation by means of suppressed recombination [16–18]. The majority of studies
on the effects of chromosome fusion and fission on speciation have been done in organisms with monocentric
chromosomes that exhibit Robertsonian translocations,
i.e. centric fusions [70–72]. These studies confirmed the
role of chromosomal fusions in reducing the frequency
of recombination. The variation in chromosome size and
number is explained as a result of frequent fusion and
fission events also in taxa with holokinetic chromosomes
[39, 73], in which kinetochores are distributed along
most of the poleward facing chromosome surface [74].
In this case, fusion is likely to behave as a stable centric
fusion and fission leads to viable chromosomal fragments that are normally inherited during meiosis [75,
76]. A recent study stressed the effect of chromosome
fusion on the recombination rate in holokinetics [77].
Moreover, studies in sedges (Carex, Cyperaceae) proved
that fusion and fission of holokinetic chromosomes also
have the potential to restrict gene flow and lead to divergence and eventually speciation [78].
The complex sex chromosome constitution revealed in
this study is likely another factor involved in the speciation of Leptidea butterflies. It has been proposed that
the Z sex chromosome could play a disproportionately
larger role in adaptive evolution compared to autosomes
[79–81]. This so-called ‘large-Z effect’ was reported in
both birds [79, 81–83] and Lepidoptera ([5, 84], the two
Šíchová et al. BMC Evolutionary Biology (2015) 15:89
largest taxa with female heterogamety. Furthermore, detailed studies on the neo-sex chromosome evolution in
geographic populations of S. cynthia and leaf-rollers of
the family Tortricidae suggest that sex chromosomal rearrangements play a major role in the formation of reproductive barriers between populations and contribute
to radiation in some lepidopteran taxa, respectively [21,
85]. In Leptidea, the multiple sex chromosome system
most likely originated by complex translocations between the ancestral WZ pair and several autosomes,
which increased the number of sex-linked genes and
thus accelerated the accumulation of genetic incompatibilities among populations. This is supported by the intraspecific stability of their multiple sex chromosomes
systems, which is in stark contrast to the evolutionary dynamics of their autosomes. Another signal of reinforcement
could be the fact that the most recently diverged sister species, L. sinapis and L. reali, display not only the largest differences in chromosome numbers in sympatry [38, 39] but
also the most different sex determination system (as shown
in this study).
Page 12 of 16
then placed into 1.5 ml Eppendorf tubes, frozen in liquid
nitrogen and stored at −80 °C until DNA extraction, except
for their genitalia which were immediately used for morphometric analysis. Hatched larvae were reared on corresponding host plants, Lathyrus pratensis for L. juvernica
and L. reali and Securigera varia for L. sinapis, at room
temperature and normal day/night regime.
Genitalia preparation and morphometric analysis
Male and female genitalia were dissected in a physiological solution and inspected under a stereomicroscope.
Lengths of two elements of the male genitalia, phallus
and saccus and one element of the female genitalia,
ductus bursae, were measured. These diagnostic characters discriminate L. sinapis from the other two species,
L. juvernica and L. reali, which cannot be reliably distinguished from each other based on morphological
features (Fig. 1; [38]).
Specimen sequencing
Conclusions
To conclude, we confirmed significant differences in the
number and structure of chromosomes within and among
closely related wood white butterflies. We showed that the
distribution of cytogenetic markers differs remarkably
even in the offspring of individual females, probably due
to irregular segregation of multivalents in meiosis. Our results suggest rapid karyotype evolution in the examined
Leptidea species and stress the role of chromosomal rearrangements, especially multiple chromosome fusions and
fissions, in their speciation. Remarkably, all three Leptidea
species have complex sex chromosome systems with 3–
4 W and 3–4 Z chromosomes. Such sex chromosome
constitutions are unique among Lepidoptera and should
be counted as an additional factor potentially contributing
to the speciation process in Leptidea butterflies. Taken together, these findings add to accumulating evidence on the
important role of chromosomal rearrangements in speciation and also point to the relevance of multiple sex chromosomes in species divergence and the formation of
reproductive barriers.
Methods
Sample collecting
Fresh adult specimens of Leptidea juvernica and L. sinapis were collected in the Czech Republic, namely in the
surroundings of České Budějovice and near Havraníky
village in the Podyjí National Park in South Moravia, respectively. The third species, L. reali, was collected in
the Montseny area near Barcelona, Spain. In the laboratory, fertilized females were kept in plastic containers to
lay eggs. The bodies of all collected individuals were
Genomic DNA (gDNA) was extracted from legs of every
female that gave progeny used in cytogenetic studies, i.e.
from 6 L. sinapis, 6 L. reali and 4 L. juvernica females,
using the NucleoSpin Tissue XS kit (Macherey-Nagel,
Düren, Germany) according to the supplier’s protocol.
To confirm the taxonomic determination of the examined specimens, molecular phylogenetic trees were constructed using one mitochondrial gene, cytochrome c
oxidase subunit 1 (COI) and one nuclear marker, the internal transcribed spacer 2 (ITS2). For each individual, a
partial sequence of both markers was amplified by polymerase chain reaction (PCR) using two pairs of primers: for
COI (658 bp) LepF1 (5′-ATTCAACCAATCATAAAGATA
TTGG-3′) and LepR1 (5′-TAAACTTCTGGATGTCCAA
AAAATCA-3′); for ITS2 (684 bp) ITS3 (5′-GCATCGATG
AAGAACGCAGC-3′) and ITS4 (5′-TCCTCCGCTTATTG
ATATGC-3′) [38].
PCR was carried out in 25-μL reaction volumes containing 1× Ex Taq buffer (TaKaRa, Otsu, Japan), 0.2 mM
dNTP mix, 5 μmol of each primer, 0.25 U Ex Taq Hot
Start DNA polymerase (TaKaRa) and about 100 ng of
template gDNA. The typical thermal cycling profile for
COI consisted of an initial denaturation period of 5 min
at 95 °C followed by 30 cycles of 30 s at 95 °C, 1 min at
44 °C and 1 min at 72 °C and by a final extension step of
7 min at 72 °C. The profile was similar for the nuclear
marker ITS2 except for the annealing temperature,
which was 50 °C. PCR products were purified using a
Wizard SV Gel and PCR Clean-Up System (Promega,
Madison, WI, USA) and sequenced using BigDye®
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).
Šíchová et al. BMC Evolutionary Biology (2015) 15:89
Phylogenetic analysis
Sequences were edited and aligned using GENEIOUS
PRO 4.7.5 created by Biomatters (http://www.geneious.com/). Our sequences were combined with all available
COI and ITS2 haplotypes of Leptidea sinapis, L. reali
and L. juvernica identified in a previous study [41] and
with sequences of L. morsei, L. amurensis, L. lactea and L.
duponcheli that were used as outgroup (Additional file 5:
Table S1). Thus, the final COI alignment contained 69
nucleotide sequences and was 658 bp long, while the
ITS2 alignment involved 28 sequences and consisted of
684 positions.
To confirm the identification of the examined specimens, neighbor-joining trees [86] were built for COI and
ITS2. Both trees were based on p-distance [87] and pairwise deletion. Node supports were assessed through 100
bootstrap replicates [88]. The trees were inferred in
MEGA6 [89].
Chromosome preparation
In each Leptidea species, two types of spread chromosome preparations were made from fifth instar male and
female larvae. Mitotic chromosomes were obtained from
wing imaginal discs characterized by a high mitotic
index [52], while meiotic chromosomes were obtained
from ovaries and testes. In both cases we used the procedure described in [45]. All preparations were passed
through a graded ethanol series (70 %, 80 % and 100 %,
1 min each) and stored at −80 °C until further use.
Preparation of polyploid nuclei
Malpighian tubules were dissected out from fifth instar
larvae of both sexes and adult females in a physiological
solution. Removed tubules were fixed in ethanol/chloroform/acetic acid (6:3:1) for 1 minute and stained in
1.5 % lactic acetic orcein. Preparations were inspected
under a light microscope for the presence of female specific sex chromatin [44].
FISH with fluorochrome-labelled probes
For the chromosome counts we used spread chromosome preparations from wing imaginal discs stained by
FISH with (TTAGG)n telomeric probes (tel-FISH), which
helped us to identify the chromosome ends. The telomeric probes were generated by non-template PCR as
described in [90] and labelled by Cy3-dUTP (GE Healthcare, Milwaukee, WI, USA) using a Nick Translation Kit
(Abbott Molecular Inc., Des Plaines, IL, USA) with
1 hour incubation at 15 °C. For tel-FISH we followed the
procedure described in [55]. The probe cocktail contained 100 ng of Cy3-labelled telomeric probe and 25 μg
of sonicated salmon sperm DNA (Sigma-Aldrich, St.
Louis, MO, USA) in 10 μl of 50 % formamide and 10 %
dextran sulfate in 2× SSC.
Page 13 of 16
GISH and CGH were used to identify the sex chromosomes and examine their molecular differentiation [43,
91]. GISH was combined with tel-FISH for better resolution of the sex chromosome constitution [55]. Genomic
DNAs for both GISH and CGH experiments were extracted separately from adult Leptidea males and females
by standard phenol-chloroform procedure. Male gDNA
was also amplified by GenomiPhi HY DNA Amplification
Kit (GE Healthcare), thereafter sonicated using a Sonopuls
HD 2070 (Bandelin Electric, Berlin, Germany) and used as
a competitor DNA [52]. The extracted male gDNA was labelled with Cy3-dUTP (GE Healthcare) and female gDNA
with fluorescein-12-dUTP (Invitrogen, Carlsbad, CA,
USA) using the Nick Translation Kit with 8 hours incubation at 15 °C.
For GISH combined with tel-FISH the probe cocktail
contained fluorescein-labelled female gDNA (300 ng),
Cy3-labelled telomeric probe (100 ng), unlabelled sonicated male gDNA (3 μg) and sonicated salmon sperm
DNA (25 μg). The probe cocktail for CGH was similar to
GISH, except that it contained Cy3-labelled male gDNA
(300 ng) instead of the telomeric probe. The preparations
were counterstained with 0.5 mg/mL DAPI and mounted
in antifade based on DABCO (Sigma-Aldrich).
FISH with biotin- and digoxigenin-labelled probes
Unlabelled 18S rDNA probe was generated by PCR from
the codling moth (Cydia pomonella) gDNA extracted
from adults by standard phenol-chloroform procedure
as described in [43]. The probe was labelled with biotin16-dUTP (Roche Diagnostics GmbH, Mannheim,
Germany) by nick translation using the Nick Translation
Kit with 1 hour and 45 minutes incubation at 15 °C.
Unlabelled H3 histone probe was obtained by PCR from
L. sinapis gDNA. PCR was carried out using degenerate
forward (5′-ATGGCNCGTACNAARCARAC-3′) and reverse (5′-TANGCACGYTCNCGGAT-3′) primers and the
final PCR product was cloned as described in [52]. The
probe was labelled in 25-μL PCR reaction containing 1×
Ex Taq buffer, 0.1 mM dATP, dGTP and dCTP, 0.065 mM
dTTP, 0.035 mM biotin-16-dUTP, 5 μmol of each M-13
universal primers, 0.25 U TaKaRa Ex Taq Hot Start DNA
polymerase and about 5 ng of plasmid DNA. The thermal
cycle profile consisted of an initial denaturation period of
2 min at 94 °C followed by 30 cycles of 30 s at 94 °C, 30 s
at 57 °C and 1 min at 72 °C and a final extension step of
2 min at 72 °C.
In FISH experiments, 18S rDNA and H3 histone
probes were combined with telomeric probes. Unlabelled
telomeric probe generated by non-template PCR (see
above) was labelled with digoxigenin (Roche Diagnostics
GmbH) using the Nick Translation Kit. The detection of
biotin was carried out as described in [43]: the signals
were detected with Cy3-conjugated streptavidin (Jackson
Šíchová et al. BMC Evolutionary Biology (2015) 15:89
ImmunoRes. Labs. Inc., West Grove, PA, USA), amplified with biotinylated anti-streptavidin (Vector Labs.
Inc., Burlingame, CA, USA) and again detected with
Cy3-conjugated streptavidin. The detection of digoxigenin was carried out by Fluorescent Antibody Enhancer
Set for DIG Detection (Roche Diagnostics GmbH). Like
in the above-mentioned FISH experiments, the preparations were counterstained with 0.5 μg/mL DAPI and
mounted in the DABCO-based antifade.
Microscopy and image processing
Preparations from FISH experiments were observed under
a Zeiss Axioplan 2 microscope (Carl Zeiss Jena, Germany).
Black-and-white images were recorded with a cooled FView CCD camera using AnalySIS software, version 3.2
(Soft Imaging System GmbH, Münster, Germany). In all
preparations, images were captured separately for each
fluorescent dye, pseudocoloured (light blue for DAPI,
green for fluorescein and red for Cy3) and superimposed
with Adobe Photoshop, version 7.0.
Additional files
Additional file 1: Figure S1. Neighbor-joining tree of nuclear ITS2
haplotypes of L. sinapis (grey background), L. reali (orange background)
and L. juvernica (blue background). Specimens sequenced and analysed
in this study are indicated by an asterisk. Leptidea amurensis, L. lactea,
L. morsei and L. duponcheli were used as outgroup. For the origin of all
specimens and GenBank accession numbers, see Additional file 5: Table
S1. The scale represents 0.01 substitutions per site. Bootstrap supports
(100 replicates) are shown next to the recovered nodes.
Additional file 2: Figure S2. The status of sex chromatin in polyploid
nuclei of three Leptidea species. The orcein-stained preparations were made
from Malpighian tubule cells of the fifth instar larvae (a, c, d) and adult
females (b). Black arrows indicate a larger deeply stained heterochromatin
body, while arrowheads show smaller bodies. (a) A lower-ploidy female
nucleus of L. sinapis with one larger and two smaller bodies. (b) A highly
polyploid female nucleus of L. sinapis with two bodies, one larger and one
smaller. (c) A male nucleus of L. reali without distinguishable heterochromatin
bodies. (d) A male nucleus of L. reali with one smaller body. Scale bar = 10 μm.
Additional file 3: Figure S3. Analysis of sex chromosome multivalents
of pachytene oocytes in Leptidea juvernica (a–d), L. sinapis (e–h) and
L. reali (i–l) using FISH with the (TTAGG)n telomeric probe. Hybridization
signals of the Cy3-dUTP-labelled telomeric probe (red) indicate
chromosome ends. Chromosomes were counterstained with DAPI (blue).
Figures (a–d), (e–h) and (i–l) show sex chromosome multivalents W1-nZ1-n:
(a, e, i) merged images of the (TTAGG)n telomeric probe and DAPI staining;
(b, f, j) DAPI images; note DAPI-highlighted heterochromatic segments of
the W chromosomes; (c, g, k) hybridization pattern of the (TTAGG)n
telomeric probe; (d, h, l) schematic drawings of the sex chromosome
multivalents; yellow dots indicate the ends of individual chromosomes
involved in the multivalents. Scale bar = 10 μm.
Additional file 4: Figure S4. Comparison of interphase nuclei sizes in
three Leptidea species. The y-axis shows the number of pixels. Micrographs of
interphase nuclei were taken from DAPI-stained spread preparations of wing
discs from three different larvae of each Leptidea species, using the same
resolution. In these micrographs, we measured the area of 144 nuclei of L.
juvernica, 154 nuclei of L. reali and 130 nuclei of L. sinapis. The measurements
were carried out using the software JMicroVision v1.2.7 [Roduit N: JMicroVision:
Image analysis toolbox for measuring and quantifying components of
high-definition images. Version 1.2.7. http://www.jmicrovision.com (accessed
27 March 2015)]. Calibration was performed using an image resolution so that
Page 14 of 16
the area of each nucleus was measured in pixels. The average size of nuclei
was calculated for each species independently and then compared between
species by one-way ANOVA using the software Statistica for Windows, version
8.0 (StatSoft, Inc., Tulsa, OK, USA). The comparison of interphase nuclei revealed
no statistically significant between-species differences in their size (F(2, 9) =
0.6782; P = 0.5425). The mean (± S.E.) area of interphase nuclei was 22434 ±
2296 pixels for L. juvernica, 19781 ± 1965 pixels for L. reali and 19835 ±
1021 pixels for L. sinapis.
Additional file 5: Table S1. List of specimens included in phylogenetic
analyses. Sequences obtained in this study are in blue, the other sequences
were downloaded from GenBank and are representative for all the COI and
ITS2 haplotypes of Leptidea sinapis, L. reali and L. juvernica identified in a
previous study [41]. The haplotype numbers correspond to those in [41].
Abbreviations
CGH: Comparative genomic hybridization; COI: Cytochrome c oxidase subunit 1;
FISH: Fluorescence in situ hybridization; gDNA: Genomic DNA; GISH: Genomic in
situ hybridization; H3: Histone H3; ITS2: Internal transcribed spacer 2; MI: Meiotic
metaphase I; NOR: Nucleolar organizer region; PCR: Polymerase chain reaction;
rDNA: Ribosomal DNA; rRNA: Ribosomal RNA; tel-FISH: Fluorescence in situ
hybridization with (TTAGG)n telomeric probe.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KS performed first cytogenetic analyses and obtained preliminary data in
Leptidea juvernica and L. sinapis. JŠ and FM conceived and designed the
experiments and collected population samples of L. juvernica and L. sinapis.
RV collected and supplied samples of the Spanish population of L. reali. JŠ
performed most experiments and analysed the data. AV and PN participated
in the preparation of FISH probes, some FISH experiments and isolation of
genes for molecular analyses. VD and RV performed phylogenetic analyses.
JŠ, PN and FM interpreted the data and drafted the manuscript. All authors
read and approved the final manuscript.
Acknowledgements
Our thanks go to Marie Korchová for excellent technical assistance and
Leonela Carabajal Paladino for help with statistical analysis. This research was
funded by Grant 14-22765S of the Czech Science Foundation, Grant
IAA600960925 of the Grant Agency of The Czech Academy of Sciences, Grant
063/2012/P of the Grant Agency of the University of South Bohemia and Grant
CGL2013-48277-P from the Spanish Ministerio de Economía y Competitividad.
JŠ and AV were supported by Grant 052/2013/P of the Grant Agency of the
University of South Bohemia, VD by a Marie Curie International Outgoing
Fellowship within the 7th European Community Framework Programme
(project no. 625997), PN by Grant 14-35819P of the Czech Science Foundation
and KS by JSPS 23380030 grant and JSPS Excellent Young Researchers Overseas
Visit Program (21–7147).
Author details
1
Institute of Entomology, Biology Centre CAS, 370 05 České Budějovice,
Czech Republic. 2Faculty of Science, University of South Bohemia, 370 05
České Budějovice, Czech Republic. 3Biodiversity Institute of Ontario,
University of Guelph, N1G 2W1 Guelph, ON, Canada. 4Institut de Biologia
Evolutiva (CSIC-Universitat Pompeu-Fabra), 08003 Barcelona, Spain.
5
Laboratory of Applied Entomology, Faculty of Agriculture, Iwate University,
Morioka 020-8550, Japan.
Received: 29 December 2014 Accepted: 7 May 2015
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